Molecular analysis of genetic recombination and DNA break repair

 描述（申请人提供）：本研究的长期目标是确定同源遗传重组和DNA断裂修复的分子机制。通过突变体的遗传分析和来自细胞的蛋白质和DNA的生物化学分析的组合来实现这一目标。这项研究使用裂殖酵母裂殖酵母以及大肠杆菌及其噬菌体λ。它们都是被广泛研究的、高度易处理的模式生物，具有包括人类在内的所有生物的共同特征。本研究主要集中在S. pombe，其高重组率有利于遗传和生物化学分析，以及细菌中重组和DNA断裂修复的主要（RecBCD）途径。 在过去成就的基础上，目前的研究重点是以下目标。1)确定减数分裂重组是如何被抑制在着丝粒内和附近，以防止染色体错误分离，不育和出生缺陷。2)确定如何选择正确的模板（姐妹或同源物）进行减数分裂DNA断裂修复，以阐明如何控制整个基因组的交换，以促进正确的减数分裂染色体分离。3)确定如何形成合适的联合DNA分子，并避免不合适的，以确保减数分裂产物（配子）的高活力。4)确定RecBCD酶与其DNA控制位点（Chi热点）相互作用的机制，以实现细菌重组的高清晰度分析。5)确定最近发现的小分子抑制剂如何阻断一种或另一种RecBCD酶活性，以开发成潜在有用的，急需的抗生素。这些目标将通过突变体的遗传分析，细胞内蛋白质和染色体位点的荧光显微镜，减数分裂细胞的DNA中间体的物理分析，以及分离蛋白质的酶和生物物理分析的组合来攻击。这些研究的结果将阐明重组和DNA断裂修复的分子机制以及对重组的控制，以确保重组发生在适当的时间和沿着染色体的位置。 减数分裂对于染色体分离、频繁出现的DNA双链断裂的无错修复以及细胞和生物多样性的产生都很重要。重组的异常可以产生染色体重排，例如易位、复制和缺失，其通常与不育、出生缺陷和癌症相关或引起不育、出生缺陷和癌症。细菌DNA断裂修复抑制剂是一种很有前途的新型抗生素，迫切需要对抗不断增加的耐药细菌的流行。

 DESCRIPTION (provided by applicant): The long-term goal of the research proposed here is to determine the molecular mechanism of homologous genetic recombination and DNA break repair. This objective is approached by a combination of genetic analysis of mutants and biochemical analysis of proteins and DNA from cells. This research uses the fission yeast Schizosaccharomyces pombe as well as the bacterium Escherichia coli and its phage lambda. All are widely studied, highly tractable model organisms with features common to all organisms, including humans. The studies are focused on meiotic recombination in S. pombe, whose high rates of recombination facilitate both genetic and biochemical analyses, and on the major (RecBCD) pathway of recombination and DNA break repair in bacteria. Building on past achievements, the research is currently focused on the following goals. 1) Determine how meiotic recombination is repressed in and near centromeres, to prevent chromosome missegregation, infertility, and birth defects. 2) Determine how the correct template (sister or homolog) is chosen for meiotic DNA break repair, to elucidate how crossing-over is controlled across the genome to favor proper meiotic chromosome segregation. 3) Determine how appropriate joint DNA molecules are formed, and inappropriate ones are avoided, to ensure high viability of meiotic products (gametes). 4) Determine the mechanism by which RecBCD enzyme interacts with its controlling site on DNA (the Chi hotspot), to allow high-definition analysis of bacterial recombination. 5) Determine how recently discovered small-molecule inhibitors block one or another RecBCD enzymatic activity, to be developed into potentially useful, sorely needed antibiotics. These goals will be attacked by a combination of genetic analysis of mutants, fluorescence microscopy of intracellular proteins and chromosomal sites, physical analysis of DNA intermediates from meiotic cells, and enzymatic and biophysical analyses of isolated proteins. The results of these studies will elucidate the molecular mechanism of recombination and DNA break repair as well as the controls on recombination that ensure that it occurs at the proper time and place along chromosomes. Recombination is important for faithful meiotic chromosome segregation, for error-free repair of frequently arising DNA double-strand breaks, and for generating cellular and organismal diversity. Aberrancies of recombination can generate chromosomal rearrangements, such as translocations, duplications, and deletions, which are often associated with or the cause of infertility, birth defects, and cancers. Inhibitors of bacterial DNA break repair are promising novel antibiotics, which are desperately needed to combat the ever-increasing prevalence of drug-resistant bacteria.